1. Technical Field
This invention relates to assays employing chromogenic reactions in which one of the reagents is a cephalosporin substrate of .beta.-lactamase.
2. Description of Related Art
.beta.-lactamases are enzymes produced by certain bacteria. These enzymes confer on these bacteria resistance to .beta.-lactam antibiotic therapy. The capacity to produce .beta.-lactamase is probably the most important and common cause of resistance to .beta.-lactam antibiotics in bacteria. For instance, if a patient infected with bacteria producing .beta.-lactamase is treated with cephalosporin, many .beta.-lactamases will recognize the cephalosporin and convert it into a metabolite with little or no antibiotic potency. Screening patient samples for .beta.-lactamase activity can avoid subjecting the patient to a course of therapy with inappropriate antibiotics.
.beta.-lactamases also have uses as a label in enzyme immunoassays (EIAs). .beta.-lactamases have very high turnover numbers, are easily available in hiqh purity from many commercial sources, have pH optima compatible with ligand-antibody binding, are relatively stable, have low molecular weight, are inexpensive, and are usually absent in body fluids.
By contrast, other commonly used enzyme labels such as alkaline phosphatase, horseradish peroxidase, and .beta.-galactosidase do not have all of these advantages in common. For instance, horseradish peroxidase requires chromogenic substrates unstable to varying degrees in the presence of hydrogen peroxide, and immunogenicity and mutagenicity of the chromogenic substrates for horseradish peroxidase are potential problems. Another disadvantage is the low yield of the horseradish peroxidase/IgG conjugation and loss of enzyme activity after conjugation. Furthermore, horseradish peroxidase exhibits markedly different stability characteristics at different pH values. Horseradish peroxidase is also difficult to use because of hemolysis problems and cross-reactivity of its substrates with hemoglobin.
.beta.-galactosidase substrates are often subjected to high rates of non-enzymatic hydrolysis leading to high numbers of failed tests. In addition, a loss of enzymatic activity upon enzyme conjugation can occur. Finally, the high molecular weight of .beta.-galactosidase presents problems in some applications. As a result, few if any commercial products exist utilizing .beta.-galactosidase.
Alkaline phosphatase is a readily available enzyme at reasonable cost. However, the efficiency of conjugation of alkaline phosphatase is rather low, typically about 5 percent, and after conjugation, only about 10 percent or less of the immunological activity of the ligand conjugated to alkaline phosphatase remains. Thermal stability of the conjugates of alkaline phosphotase is typically very low. Furthermore, because many samples from humans contain alkaline phosphatase, it is difficult to detect the labelled alkaline phosphatase from the endogenous alkaline phosphatase activity.